Our previous linkage study indicated that a gene on chromosome 4q contributes to insulin action in Pimas. A candidate gene in this region is FABP2 which encodes the intestinal fatty acid binding protein (IFABP). To determine whether FABP2 has a role in insulin action, we analyzed the FABP2 coding sequences and detected a single base substitution in exon II that would result in an alanine (Ala) to threonine (Thr) substitution at position 54 of the protein. Pimas with a Thr-encoding FABP2 allele (frequency 0.3) were found to have increased insulin resistance and increased fat oxidation rates as compared to Pimas with an Ala-encoding FABP2 allele (frequency 0.7). To analyze these IFABP proteins biochemically for functional variation that could result in the clinical variation seen in the Pimas, we isolated the cDNA for the Ala form of IFABP and produced the cDNA for the Thr form by site-directed mutagenesis. Both cDNAs were expressed in E. coli and their products purified to homogeneity. Three-dimensional structural variation between the two proteins is currently being analyzed by X-ray crystallography. Functional variation was observed in the two recombinant proteins' affinity for long chain fatty acids, where the Thr form binds twice as tightly as the Ala form in titration microcalorimetry studies. The cDNA for each protein was also permanently transfected into enterocyte-like tissue culture cells. Upon exposure to radiolabeled fatty acids, cells expressing the Thr protein processed fatty acids twice as quickly as cells expressing the Ala protein. Since fatty acid bound to IFABP interacts with long chain acyl CoA synthetase to form of acyl CoA, we will analyze the interaction of both forms of IFABP with acyl CoA synthetase. To this end, we have isolated the human intestinal acyl CoA synthetase cDNA and expressed this protein in yeast.